Plant pyruvate, orthophosphate dikinase (PPDK; EC2.7.9.1) is one of the important enzymes constituting the C4 cycle and it has been believed that there is a high correlation between PPDK activity and photosynthesis rate in C4 plants. Further, PPDK has the lowest activity among enzymes constituting the C4 cycle; the PPDK reaction has been regarded as a rate-limiting stage of C4 photosynthesis. Also, PPDK is a tetramer composed of four subunits which are weakly associated with each other. When exposed to low temperature conditions (at or below 12° C.), PPDK is known to be dissociated into dimers or monomers, thus rapidly losing its activity. In general, maize PPDK will lose approximately 70% of its activity when treated at 0° C. for 20 minutes. Meanwhile, the activity of maize PPDK was measured at various temperatures to prepare an Arrhenius plot, indicating that there was an inflection point at 11.7° C., which was found to match the critical temperature for maize growth. In view of these points, a decrease in PPDK activity has been regarded as the main factor responsible for slowdown of photosynthesis in C4 plants at low temperature.
Flaveria brownii (F. brownii), an Asteraceae plant, is categorized as a C3/C4 intermediate type and its PPDK is known to be hardly deactivated even when treated at a temperature as low as 0° C. (Burnell J N, A comparative sturdy of the cold-sensitivity of pyruvate, Pi dikinese in Flaveria species, Plant Cell Physiol., 31:295-297 (1990)). Hence, it was expected that this cold-tolerant PPDK gene could be used to create C4 plants capable of C4 photosynthesis at a lower temperature, i.e., C4 plants more resistant to cold.
In the previous studies, the inventors of the present invention succeeded in determining a region important for cold tolerance of PPDK by isolation and DNA sequencing of the F. brownii PPDK gene. They also demonstrated that the sequence of this region could be used to convert cold-sensitive PPDK into a cold-tolerant form through recombination between this sequence and the DNA of cold-sensitive PPDK derived from other plant (WO95/15385; Usami S, Ohta S, Komari T, Burnell J N, Cold stability of pyruvate, orthophosphate dikinase of Flaveria brownii, Plant Mol Biol, 27:969-80 (1995); Ohta S, Usami S, Ueki J, Kumashiro T, Komari T, Burnell J N, Identification of the amino acid residues responsible for cold tolerance in Flaveria brownii pyruvate, orthophosphate dikinase, FEBS Lett, 396:152-6 (1996)).
Through many studies of maize transformants, however, the inventors of the present invention found that effects of the artificially introduced PPDK (introduced PPDK) were masked by naturally-occurring PPDK in C4 plants (endogenous PPDK). This would be attributed to an abundance of endogenous PPDK constituting several percent of soluble proteins in C4 plants. Further, the inventors of the present invention found that heterotetramers could be formed between introduced PPDK subunits and endogenous PPDK subunits.
To overcome the phenomenon where effects of the introduced PPDK are masked by the endogenous PPDK, two techniques are available, one of which involves increasing the expression level of the introduced PPDK and the other is inhibition of the endogenous PPDK. The former involves integrating a sequence (e.g., intron) into a gene construct for transformation (in most cases, an intron(s) being integrated between a promoter and a structural gene) to increase the expression level of an externally introduced gene (WO96/30510: PLD intron, WO97/47755: double-ligated introns). The latter is an antisense technique for inhibiting the expression level of an object gene by introduction of a gene whose mRNA has a sequence complementary to mRNA from the object gene to be inhibited from expression (Japanese Patent No. 2651442 and its divisional patent No. 2694924). The inventors of the present invention have tried these techniques.
In increasing the expression level of the introduced PPDK by integration of an intron(s) etc., a gene construct used for transformation was constructed in a general manner through ligation between a maize PPDK promoter (Glackin Calif., Grula J W, Organ-specific transcripts of different size and abundance derive from the same pyruvate, orthophosphate dikinase gene in maize, PNAS, 87:3004-3008 (1990)) and a cDNA molecule of the PPDK gene derived from F. brownii, F. bidentis (Usami S, Ohta S, Komari T, Burnell J N, Cold stability of pyruvate, orthophosphate dikinase of Flaveria brownii, Plant Mol Biol, 27:969-80 (1995)) or maize (Matsuoka M, Structure, genetic mapping, and expression of the gene for pyruvate, orthophosphate dikinase from maize, J. Biol. Chem, 265:16772-16777 (1990)). At the same time, the inventors of the present invention attempted to increase the expression level of the introduced PPDK by inserting any one of the following introns between the promoter and the structural gene: Intron 1 of the Castor bean catalase gene (Ohta S, Mita S, Hattori T, Nakamura K, Construction and expression in tobacco of a β-glucuronidase (GUS) reporter gene containing an intron within the coding sequence, Plant Cell Physiol, 31:805-813 (1990)), Intron 1 of the rice phospholipase D gene (Ueki J, Morioka S, Komari T, Kumashiro T, Purification and characterization of phospholipase D from rice and maize (Zea mays L.), Plant Cell Physiol, 36:903-914 (1995)), Intron 1 of maize ubiquitin (Christensen et al., (1992)) and Intron 1 of maize Shrunken-1 (Vasil V, Clancy M, Ferl R J, Vasil I K, Hannah L C, Increased gene expression by the first intron of maize Shrunken-1 locus in grass species, Plant Physiol, 91:1575-1579 (1989)). Further, based upon a report suggesting that repeated introns resulted in an increased expression level (Ueki J, Ohta S, Morioka S, Komari T, Kuwata S, Kubo T, Imaseki H, The synergistic effects of two-intron insertions on heterologous gene expression and advantages of the first intron of a rice gene for phospholipase D, Plant Cell Physiol, 40:618-623 (1999)), the inventors of the present invention also attempted to insert multiple repeated introns.